Tehran University of Medical Sciences Iranian Journal of Parasitology 1735-7020 14 4 2019 12 29 584 591 EN Mohammad Hossein FEIZ HADDAD Infectious and Tropical Diseases Research Center, Health Research Institute, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran AND Department of Parasitology, Faculty of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran Saeed KHOSHNOOD Student Research Committee, School of Medicine, Bam University of Medical Sciences, Bam, Iran Mohammad Reza MAHMOUDI Molecular and Cellular Research Center, Faculty of Medicine, Guilan University of Medical Sciences, Rasht, Iran Habib HABIBPOUR Department of Parasitology, Faculty of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran AND Student Research Committee, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran Selman A. ALI Interdisciplinary Biomedical Research Centre, School of Science and Technology, Nottingham Trent University, Nottingham, UK Habibollah MIRZAEI Hepatitis Research Center, Lorestan University of Medical Sciences, Khorramabad, Iran AND Department of Virology, Faculty of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran Rezvan FEIZ HADDAD Department of Midwifery, Faculty of Medicine, Dezful University of Medical Sciences, Dezful, Iran Kambiz AHMADIANGALI Department of Bio-Statistics, Faculty of Health, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran 2018 08 21 2019 12 29 Background: This study was conducted to determine the presence and molecular identify of Acanthamoeba, Naegleria and Vermamoeba in unimproved hot springs. Methods: From Jul to Aug 2017, 54 water samples were collected from hot springs in different parts of the Guilan Province, North Iran. For the isolation of Acanthamoeba, Naegleria and Vermamoeba approximately 500 ml of the water samples were filtered through a cellulose nitrate membrane with a pore size of 0.45 μm. The filter was transferred onto non-nutrient agar plates seeded with Gram-negative bacteria (Escherichia coli) as a food source. The morphological key of page was used to identify free‐living amoebae (FLA) using an inverted microscope, PCR amplification targeting specific genes for each genus and sequencing determined frequent species and genotypes base on NCBI database. Results: Fifteen of the 54 samples were positive by culture and/or PCR for Acanthamoeba and other FLA from unimproved hot springs. By sequencing the positive isolates, the strains were shown to belong to Acanthamoeba castellanii (12 case isolates belonged to T4 genotype), 4 cases of V. vermiformis, and 3 cases of N. australiensis, 2 cases of N. pagei and 1 cases of N. gruberi. Conclusion: Although FLA-mediated illnesses are not as high as in environmental distribution, but because of a poor prognosis, more investigations about FLA distribution in hot springs is critical. Hot spring may enhance exposure of the amoebae in individuals. Hence, more attention to unimproved hot springs is needed to prevent free-living amoebae mediated diseases. https://ijpa.tums.ac.ir/index.php/ijpa/article/view/2284